Alzheimer's disease (AD) is a long-term, debilitating disorder that causes loss of memory and cognitive functions. There is an urgent need for effective therapeutic agents to ameliorate the symptoms of AD. Important molecular drug targets in AD are the gamma-secretases that produce the neurotoxic Abeta peptides in AD. The regulated secretory pathway of neurons represents the major source of secreted Abeta peptides that accumulate in extracellular amyloid plaques in AD. However, previous screens for inhibitors of gamma-secretase have only analyzed the minor constitutive secretory pathway for Abeta peptide production. Different proteases are present in the regulated secretory pathway compared to the constitutive secretory pathway. Clearly, gamma-secretase in the regulated secretory pathway must be targeted for drug inhibition to provide the greatest reduction of Abeta. Therefore, the Phase I project developed high-throughput assays for gamma-secretases in regulated secretory vesicles that produce Abeta. A candidate inhibitor of gamma-secretases was identified, which will provide the basis for design and synthesis of 'focused' and 'optimized' libraries for this Phase II project. Phase I results also indicate feasibility to find agents that selectively inhibit gamma-secretases-42 compared to gamma-secretases-40. The Phase I project also developed neuronal chromaffin cell and brain cortical neuron assays for Abeta in the regulated secretory pathway. The goal of this phase II project will be to utilize the regulated secretory vesicle as the major site and target of Abeta peptide production in neuronal cells, for identifying inhibitor molecules that selectively inhibit gamma-secretases-42 compared to gamma-secretases-40. Such inhibitors will be considered as lead compounds. In specific aim 1, efforts for 'focused' library compounds will be screened in the high throughput in vitro assays to identify inhibitors of gamma-secretases-40 and gamma-secretases-42 in chromaffin vesicles; inhibitors will then be tested in chromaffin cells for reduction of Abeta. In the second aim, further 'optimized' libraries will be designed and synthesized based on structural features of inhibitors from the 'focused' library, screened in the high throughput assays, and tested for reduction of Abeta in the regulated secretory pathway of neuronal chromaffin cells. In the third aim, lead compounds will undergo evaluation in brain neuronal cells to identify compounds that reduce production of Abeta peptides in the regulated secretory pathway. Results will likely identify novel lead compounds for future pre-clinical animal studies for development of effective drugs for AD.